Use of a nuclease inhibitor or interleukin-10 (IL-10) for the preparation of a therapeutic composition for improving transfection of a polynucleotide into a cell and compositions useful in gene therapy

ABSTRACT

Described is the use of a nuclease inhibitor or of interleukin-10 (IL-10) for the preparation of a therapeutic composition for improving transfection of a polynucleotide into a cell, and to compositions comprising a mixture of polynucleotide and nuclease inhibitor and/or interleukin-10.

[0001] The present invention relates to the use of a nuclease inhibitoror of interleukin-10 (IL-10) for the preparation of a therapeuticcomposition for improving transfection of a polynucleotide into a cell,and to compositions comprising a mixture of polynucleotide and nucleaseinhibitor and/or interleukin-10. Such a composition is useful in genetherapy, vaccination, and any therapeutic situation in which agene-based product is administered to cells in vivo.

[0002] Gene therapy has generally been conceived as principallyapplicable to heritable deficiency diseases (cystic fibrosis,dystrophies, haemophilias, etc.) where permanent cure may be effected byintroducing a functional gene. However, a much larger group of diseases,notably acquired diseases (cancer, AIDS, multiple sclerosis, etc.) mightbe treatable by transiently engineering host cells to produce beneficialproteins.

[0003] Applications are, for example, the treatment of musculardystrophies or of cystic fibrosis. The genes of Duchenne/Becker musculardystrophy and cystic fibrosis have been identified and encodepolypeptides termed dystrophin and cystic fibrosis transmembraneconductance regulator (CFTR), respectively. Direct expression of thesegenes within, respectively, the muscle or lung cells of patients shouldcontribute to a significant amelioration of the symptoms by expressionof the functional polypeptide in targeted tissues. Moreover, studies incystic fibrosis have suggested that one would need to achieve expressionof the CFTR gene product in only about 5% of lung epithelial cells inorder to significantly improve the pulmonary symptoms.

[0004] Another application of gene therapy is vaccination. In thisregard, the immunogenic product encoded by the polynucleotide introducedin cells of a vertebrate may be expressed and secreted or be presentedby said cells in the context of the major histocompatibility antigens,thereby eliciting an immune response against the expressed immunogen.Functional polynucleotides can be introduced into cells by a variety oftechniques resulting in either transient expression of the gene ofinterest, referred to as transient transfection, or permanenttransformation of the host cells resulting from incorporation of thepolynucleotide into the host genome.

[0005] Successful gene therapy depends on the efficient delivery to andexpression of genetic information within the cells of a living organism.Most delivery mechanisms used to date involve viral vectors, especiallyadeno- and retroviral vectors. Viruses have developed diverse and highlysophisticated mechanisms to achieve this goal including crossing of thecellular membrane, escape from lysosomal degradation, delivery of theirgenome to the nucleus. Consequently, viruses have been used in many genedelivery applications in vaccination or gene therapy applied to humans.The use of viruses suffers from a number of disadvantages: retroviralvectors cannot accommodate large-sized DNA (for example, the dystrophingene which is around 13 Kb), the retroviral genome is integrated intohost cell DNA and may thus cause genetic changes in the recipient celland infectious viral particles could disseminate in the organism or inthe environment and adenoviral vectors can induce a strong immuneresponse in treated patients (Mc Coy et al., Human Gene Therapy 6(1995), 1553-1560; Yang et al., Immunity 1 (1996), 433-442).Nevertheless, despite these drawbacks, viral vectors are currently themost useful delivery systems because of their efficiency. Non-viraldelivery systems have been developed which are based onreceptor-mediated mechanisms (Perales et al., Eur. J. Biochem. 226(1994), 255-266; Wagner et al., Advanced Drug Delivery Reviews 14(1994), 113-135), on polymer-mediated transfection such aspolyamidoamine (Haensler and Szoka, Bioconjugate Chem. 4 (1993),372-379), dendritic polymer (WO 95/24221), polyethylene imine orpolypropylene imine (WO 96/02655), polylysine (U.S. Pat. No. 5,595,897or FR 2 719 316) or on lipid-mediated transfection (Felgner et al.,Nature 337 (1989), 387-388) such as DOTMA (Felgner et al., Proc. Natl.Acad. Sci. USA 84 (1987), 7413-7417), DOGS or Transfectam™ (Behr et al.,Proc. Natl. Acad. Sci. USA 86 (1989), 6982-6986), DMRIE or DORIE(Felgner et al., Methods 5 (1993), 67-75), DC-CHOL (Gao and Huang, BBRC179 (1991), 280-285), DOTAP™ (McLachlan et al., Gene Therapy 2 (1995),674-622) or Lipofectamine™. These systems present potential advantageswith respect to large-scale production, safety, targeting oftransfectable cells, low immunogenicity and the capacity to deliverlarge fragments of DNA. Nevertheless their efficiency in vivo is stilllimited.

[0006] Finally, in 1990, Wolff et al. (Science 247 (1990), 1465-1468)have shown that injection of naked RNA or DNA, without a specialdelivery system, directly into mouse skeletal muscle results inexpression of reporter genes within the muscle cells. This technique fortransfecting cells offers the advantage of simplicity and experimentshave been conducted that support the usefulness of this system for thedelivery to the lung (Tsan et al., Am. J. Physiol. 268 (1995),L1052-L1056; Meyer et al., Gene Therapy 2 (1995), 450460), brain(Schwartz et al., Gene Therapy 3 (1996), 405411), joints (Evans andRoddins, Gene therapy for arthritis; In Wolff (ed) Gene therapeutics:Methods and Applications of direct Gene Transfer. Birkhaiser. Boston(1990), 320-343), thyroid (Sikes et al., Human Gen. Ther. 5 (1994),837-844), skin (Raz et al., Proc. Natl. Acad. Sci. USA 91 (1994),9519-9523) and liver (Hickman et al., Hum. Gene Ther. 5 (1994),1477-1483). Nevertheless, Davis et al. (Human Gene Therapy 4 (1993),151-159 and Human Mol. Genet. 4 (1993), 733-740) observed a largevariability of expression due to nonuniform distribution of naked DNAinjected into skeletal muscle in vivo. Only a small proportion of themuscle fibers (about 1-2%) are transfected and this level of genetransfer would be insufficient for the treatment of primary myopathies.The authors propose solutions in order to obtain an improvement of theefficiency of gene transfer (resulting in about 10% of transfectedmuscle fibers) by preinjecting muscles with a relatively large volume ofhypertonic sucrose or with toxins, for example cardiotoxin isolated fromsnake, in order to stimulate regeneration of muscles. These methods,although promising, would not be applicable for human treatment.

[0007] Thus, the available delivery methods are not satisfactory interms of safety or efficiency for their implementation in in vivo genetherapy.

[0008] Therefore, the technical problem underlying the present inventionis the provision of improved methods and means for the delivery ofnucleic acid molecules in gene therapy.

[0009] This technical problem is solved by the provision of theembodiments as defined in the claims.

[0010] Accordingly, the present invention relates to the use of anuclease inhibitor for the preparation of a therapeutic composition forintroducing a polynucleotide into a cell. It was surprisingly found thatthe addition of a nuclease inhibitor when transfecting a polynucleotideinto vertebrate tissue leads to a dramatic improvement of thetransfection efficiency. In particular, it was surprisingly found thatif the polynucleotide is injected together with a nuclease inhibitor,e.g., into muscular tissue, the transfection is not only improved in thesurrounding of the injection site but also in other areas of the muscle.Thus, the present invention preferably relates to the use of a nucleaseinhibitor for the preparation of a pharmaceutical composition for animproved introduction of a polynucleotide into a cell. The term“improved introduction” in the scope of the present invention means, inthis regard, a more efficient uptake of a polynucleotide by cells when anuclease inhibitor is present compared to an introduction performedwithout a nuclease inhibitor. This can be determined by comparing theamount of the polynucleotide taken up without the use of a nucleaseinhibitor and comparing this amount with the amount taken up by thecells when using a nuclease inhibitor under the same experimentalconditions. Preferably, the improved introduction can be determined by ahigher amount of expression of the polynucleotide transferred into thecells when using a nuclease inhibitor in comparison to a situation whereno nuclease inhibitor is used.

[0011] Preferably, an improved introduction of the polynucleotide intothe cell means that the uptake of the polynucleotide by cells is notonly improved at the site of administration of the polynucleotide andnuclease inhibitor but is also improved in neighboring cells.Particularly preferred, an improved introduction means that the usednuclease inhibitor shows the same improving effect on the uptake of apolynucleotide by cells as does G-actin when compared to theadministration of the polynucleotide without any nuclease inhibitor.

[0012] The therapeutic compositions according to the first aspect of thepresent invention are particularly useful for the delivery ofpolynucleotides to cells or tissues of a subject in the scope of a genetherapeutic method but are not limited to such use. The term “genetherapy method” is preferably understood as a method for theintroduction of a polynucleotide into cells either in vivo or byintroduction into cells in vitro followed by re-implantation into asubject. “Gene therapy” in particular concerns the case where the geneproduct is expressed in a target tissue as well as the case where thegene product is excreted, especially into the blood stream. In the scopeof the present invention the term “introduction” means the transfer ofthe polynucleotide into a cell (transfection).

[0013] In the scope of the present invention the term “nuclease” meansan enzyme with the capability to degrade nucleic acid molecules. Suchnucleases encompass nucleases which can degrade single stranded nucleicacid molecules as well as nucleases which can degrade double strandednucleic acid molecules. Furthermore, the nuclease can have thecapability to degrade RNA or DNA. Preferably, it is a nuclease whichdegrades DNA. More preferably, the nuclease is a DNAse I, andparticularly preferred a human nuclease.

[0014] A DNAse I in the scope of the present invention is to beunderstood as an endonuclease that hydrolyzes double-stranded orsingle-stranded DNA preferentially at sites adjacent to pyrimidinenucleotides. The product of this hydrolysis is a complex mixture of5′-phosphate mono- and oligonucleotides. In the presence of Mg²⁺, aDNAse I attacks each strand of DNA independently and the sites ofcleavage are distributed in a statistically random fashion. Furthermore,in the presence of Mn²⁺, DNAse I cleaves both strands of DNA atapproximately the same site to yield fragments of DNA that areblunt-ended or that have protruding termini only one or two nucleotidesin length.

[0015] In the scope of the present invention a nuclease inhibitor isdefined by its capacity to act on a nuclease activity in a way thatleads to a total or partial loss of the property of the nuclease todegrade a nucleic acid molecule. This capacity can be determined byincubating the potential inhibitor with the nuclease and with a nucleicacid molecule, which is normally degraded by the nuclease, underconditions which normally allow the nuclease to degrade the nucleic acidmolecule and by determining whether the inhibitor represses or decreasesthe degradation of the nucleic acid molecule. The inhibitor can bind tothe nuclease or can react with it. The inhibitor can be, for example, achemical compound or a protein or fragment of a protein having nucleaseinhibitor activity. Examples are antibodies or parts of antibodies whichreact specifically with a nuclease. Preferably, such an antibody is amonoclonal antibody.

[0016] It is possible for the person skilled in the art to find in theliterature molecules described as nuclease inhibitors. Described are,for example, antibiotic compounds such as coumermycin or novobiocin,nalidixic or oxolinic acids (Fox and Studzinski, J. Histochem Cytochem.30 (1982), 364-370), ciprofloxacin (CFL) or norfloxacin (Tempel andIgnatius, Arzneimittelforschung 42 (1992), 1031-1036) andaurintricarboxylic acid (ATA) (Benchokroum et al., Biochem. Pharmacol.49 (1995), 305-313). More preferably, the nuclease inhibitor is not anacidic molecule and, if it is an acidic molecule, it is administered ina buffered neutral solution. In a preferred embodiment the nucleaseinhibitor is an inhibitor of a DNAse I. More preferably, it is apolypeptide or a fragment of a polypeptide which inhibits a DNAse I. Ina particularly preferred embodiment the nuclease inhibitor is theglobular form of actin (G-actin) (Harwell et al., J. Biol. Chem. 255(1980), 1210-1220). Several publications have described the ability ofG-actin to interact with a large number of actin binding proteinsincluding DNAse I (Sheterline and Sparrow, Protein Profile 1 (1994),1-121). G-actin binds DNAse I with high affinity and is a potentinhibitor (K_(i) 1 nM) of DNA hydrolytic activity (Lacks, J. Biol. Chem.256 (1981), 2644-2648; Pinder and Gratzer, Biochemistry 21 (1982),4886-4890). Based on these observations, Snabes et al. (J. Biol. Chem.256 (1981), 6291-6295) have developed an immunoprecipitation assay basedon DNAse I/actin binding using rabbit skeletal muscle actin or actinpresent in tissue and cell extracts. It is known that the interfacebetween DNAse I and G-actin involves two exposed loops in subdomain 11(residues P38 to S52) and IV (residues T194 to T203). Thus, a fragmentof G-actin used in the scope of the present invention preferablycomprises the residues forming these loops. The G-actin may be anaturally occurring form of G-actin, a modified G-actin (Carlier,Biochemistry 31 (1992), 300-309), a polypeptide complexed form (Peitschet al., EMBO J. 12 (1993), 371-377) or a truncated form as long as theresulting polypeptide retains its ability to inhibit DNAse I activity.The G-actin may be, in principle, of any origin, preferably fromvertebrates, more preferably from mammals, e.g. porcine, rabbit, bovineor human origin. G-actin is an ubiquitously expressed polypeptide andcan be purified from mainly skeletal muscle and heart or produced byrecombinant technology. G-actin is supplied for example by Sigma. Theterm nuclease inhibitor as used herein also means a nuclease inhibitoras derived from a variety of mammalian species, including, for example,human, simian, rabbit, bovine, porcine or murine. A nuclease inhibitorcan be produced, for example, by recombinant technology.

[0017] G-actin is known to have further activities, e.g. the capacity tobind divalent metal ions, such as calcium and magnesium ions, and thecapacity to bind and hydrolyze ATP, which may cause or contribute to theobserved effect of improved introduction of a polynucleotide into cells.Thus, also other proteins having these properties might be useful in thescope of the present invention.

[0018] In a preferred embodiment of the use according to the firstaspect of the present invention, the prepared therapeutic composition isin a form for administration into a vertebrate tissue. These tissuesinclude those of muscle, skin, brain, lung, liver, spleen, bone marrow,thymus, heart, lymph, bone, cartilage, pancreas, kidney, gall bladder,stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye,gland, connective tissue, blood, tumor etc. Cells where the improvedtransfection of a foreign polynucleotide would be obtained are thosefound in each of the listed target tissues (muscular cells, airwaycells, hematopoetic cells, etc.). The administration may be made byintradermal, subdermal, intravenous, intramuscular, intranasal,intracerebral, intratracheal, intraarterial, intraperitoneal,intravesical, intrapleural, intracoronary or intratumoral injection,with a syringe or other devices.

[0019] Transdermal administration is also contemplated, as areinhalation or aerosol administration.

[0020] In a preferred embodiment, the therapeutic composition is for theintroduction into muscle tissue, more preferably, by intramuscularinjection routes.

[0021] In a preferred embodiment of the first aspect of the presentinvention, the use of a nuclease inhibitor for the preparation of atherapeutic composition for improving transfection of a polynucleotideinto a cell is provided wherein said therapeutic composition isadministered independently from a second administration consisting inadministration of a composition containing at least one polynucleotide.According to the present invention, the first administration can be doneprior to, concurrently with or subsequent to the second administration,and vice-versa. The therapeutic composition administration and secondadministration can be performed by different delivery routes (systemicdelivery and targeted delivery, or targeted deliveries for example). Ina preferred embodiment, each should be done into the same target tissueand most preferably by injection.

[0022] In a further embodiment of the use according to the presentinvention, the therapeutic composition further comprises at least onepolynucleotide. In a particularly preferred embodiment, thepolynucleotide which is contained in the composition, contains and iscapable of functionally expressing a gene in said cell.

[0023] The polynucleotide may be a DNA or RNA, single or doublestranded, linear or circular, natural or synthetic, modified or not (seeU.S. Pat. No. 5,525,711, U.S. Pat. No. 4,711,955 or EP-A 302 175 formodification examples). It may be, inter alia, a genomic DNA, a cDNA, anmRNA, an antisense RNA, a ribosomal RNA, a ribozyme, a transfer RNA orDNA encoding such RNAs. “Polynucleotides” and “nucleic acids” aresynonyms with regard to the present invention. The polynucleotide mayalso be in the form of a plasmid or linear polynucleotide which containsat least one expressible sequence of nucleic acid that can generate apolypeptide, a ribozyme, an antisense RNA or another molecule ofinterest upon delivery to a cell. The polynucleotide can also be anoligonucleotide which is to be delivered to the cell, e.g., forantisense or ribozyme functions. The polynucleotide according to thepresent invention should preferably be understood as a nakedpolynucleotide (Wolff et al., Science 247 (1990), 1465-1468) or as apolynucleotide associated or complexed with a viral polypeptide or acationic compound or with any component which can participate in theuptake of the polynucleotide into the cells (see Ledley, Human GeneTherapy 6 (1995), 1129-1144 for a review). Both DNA or RNA can bedelivered to cells to form therein a polypeptide of interest.Preferably, the polynucleotide present in the therapeutic composition isin the form of plasmid DNA. If the polynucleotide contains the propergenetic information, it will direct the synthesis of relatively largeamounts of the encoded polypeptide. When the polynucleotide delivered tothe cells encodes an immunizing polypeptide, the use according to theinvention can be applied to achieve improved and effective immunityagainst infectious agents, including intracellular viruses, and alsoagainst tumor cells. The genetic information necessary for expression bya target cell comprise all the elements required for transcription ofsaid DNA into mRNA and for translation of mRNA into polypeptide.Transcriptional promoters suitable for use in various vertebrate systemsare well known. For example, suitable promoters include viral promoterslike RSV, MPSV, SV40, CMV or 7.5 k, vaccinia promoter, induciblepromoters, etc. The polynucleotide can also include intron sequences,targeting sequences, transport sequences, sequences involved inreplication or integration. Said sequences have been reported in theliterature and can be readily obtained by those skilled in the art. Thepolynucleotide can also be modified in order to be stabilized withspecific components as spermine.

[0024] According to the invention, the polynucleotide can be homologousor heterologous to the target cells into which it is introduced.Advantageously said polynucleotide encodes all or part of a polypeptide,especially a therapeutic or prophylactic polypeptide. A polypeptide isunderstood to be any translational product of a polynucleotideregardless of size, and whether glycosylated or not, and includespeptides and proteins. Therapeutic polypeptides include as a primaryexample those polypeptides that can compensate for defective ordeficient proteins in an animal or human organism, or those that actthrough toxic effects to limit or remove harmful cells from the body.They can also be immunity conferring polypeptides which act asendogenous immunogens to provoke a humoral or cellular response, orboth. Examples of polypeptides encoded by the polynucleotide areenzymes, hormones, cytokines, membrane receptors, structuralpolypeptides, transport polypeptides, adhesines, ligands, transcriptionfactors, traduction factors, replication factors, stabilization factors,antibodies, more especially CFTR, dystrophin, factors VIII or IX, E6 orE7 from HPV, MUC1, BRCA1, interferons, interleukin (IL)2, IL-4, IL-6,IL-7, IL-12, GM-CSF (Granulocyte Macrophage Colony Stimulating Factor),the tk gene from Herpes Simplex type 1 virus (HSV-1), p53 or VEGF. Thepolynucleotide can also code for an antibody. In this regard, antibodyencompasses whole immunoglobulins of any class, chimeric antibodies andhybrid antibodies with dual or multiple antigen or epitopespecificities, and fragments, such as F(ab)₂, Fab′, Fab including hybridfragments and anti-idiotypes (U.S. Pat. No. 4,699,880).

[0025] Furthermore, the invention relates to a composition for theintroduction of a polynucleotide into a cell, said compositioncomprising at least one polynucleotide and at least one nucleaseinhibitor. Polynucleotide and nuclease inhibitor components are definedas above.

[0026] In a preferred embodiment, the nuclease inhibitor contained insaid composition is a DNAse inhibitor and even more preferred, a DNAse Iinhibitor. In a particularly preferred embodiment, the nucleaseinhibitor is G-actin or a fragment thereof, having the capability toinhibit a DNAse I.

[0027] The amount of nuclease inhibitor in the compositions rangespreferably between 4×10⁻⁵ and 4 μg per μg of DNA, preferably between4×10⁻⁴ and 2 μg per μg of DNA. In a preferred embodiment, saidcomposition comprises between 4×10⁻³ and 4×10⁻¹ μg of nuclease inhibitorper μg of DNA.

[0028] In another preferred embodiment, the polynucleotide which iscontained in the composition, contains and is capable of functionallyexpressing, a gene in a cell, preferably in a vertebrate cell. Oneparticularly preferred embodiment of the invention is a compositionwherein said polynucleotide is naked. Nevertheless, the polynucleotidecomprised in said composition can also be associated with viralpolypeptides, or complexed with cationic components, more particularlywith cationic lipids. In general, the concentration of polynucleotide inthe composition is from about 0.1 μg/ml to about 20 mg/ml.

[0029] In a further preferred embodiment the composition furthercomprises at least one component selected from the group consisting ofchloroquine, protic compounds such as propylene glycol, polyethyleneglycol, glycerol, ethanol, 1-methyl L-2-pyrrolidone or derivativesthereof, aprotic compounds such as dimethylsulfoxide (DMSO),diethylsulfoxide, di-n-propylsulfoxide, dimethylsulfone, sulfolane,dimethyl-formamide, dimethylacetamide, tetramethylurea, acetonitrile orderivatives. The composition may also advantageously comprise a sourceof a cytokine which is incorporated in the form of a polypeptide or as apolynucleotide encoding the cytokine. Preferably, said cytokine isinterleukin 10 (IL-10). According to a preferred embodiment, thecomposition comprises 5-15% of DMSO and/or 0.001 to 1 μg preferably 0.01to 0.1 μg of IL-10.

[0030] In a further preferred embodiment the composition according tothe first aspect of the invention can be used in a method for thetherapeutic treatment of humans or animals. In this particular case, thecomposition according to the invention may also comprise apharmaceutically acceptable injectable carrier (for examples, seeRemington's Pharmaceutical Sciences, ¹⁶th ed. 1980, Mack PublishingCo.). The carrier is preferably isotonic, hypotonic or weakly hypertonicand has a relatively low ionic strength, such as provided by a sucrosesolution. Furthermore, it may contain any relevant solvents, aqueous orpartly aqueous liquid carriers comprising sterile, pyrogen-free water,dispersion media, coatings, and equivalents or diluents (e.g. Tris-HCL,acetate, phosphate), emulsifiers, solubilizers or adjuvants. The pH ofthe pharmaceutical preparation is suitably adjusted and buffered.

[0031] Further, the present invention also relates to a process forintroducing a polynucleotide into cells wherein said process comprisescontacting said cells with at least one composition according to theinvention. This process may be applied by direct administration of saidcomposition to cells of the animal in vivo, or by in vitro treatment ofcells which can be extracted from the animal and then re-introduced intothe animal body (ex vivo process). According to the practice of theinvention, targeted “cells” and “in vivo administration route” aredefined as above described.

[0032] The present invention also relates to a process for introducing apolynucleotide into cells wherein said process comprises contacting thecells with said polynucleotide prior to, concurrent with or subsequentto contacting them with a nuclease inhibitor. Preferably, the cells arefirst contacted with the nuclease inhibitor and afterwards with thepolynucleotide. “Nuclease inhibitor”, “polynucleotide” and the targetcells are defined as above.

[0033] Preferably, muscle is used as a site for the delivery andexpression of a polynucleotide in a number of therapeutic applicationsbecause animals have a proportionately large muscle mass which isconveniently accessed by direct injection through the skin. Accordingly,in a preferred case, the invention concerns a process for introducing apolynucleotide, preferably in naked form, into muscle cells in vivo,comprising the steps of administering in vivo at least a polynucleotideand at least a nuclease inhibitor, preferably G-actin, preferablyintramuscularly, whereby the polynucleotide is introduced into musclecells of the tissue. The polynucleotide may encode a therapeuticpolypeptide that is expressed by the muscle cells and eventuallysecreted into the blood stream after the contacting step to providetherapy to the vertebrate. Similarly, it may encode an immunogenicpolypeptide that is expressed by the muscle cells after the contactingstep and which generates an immune response, thereby immunizing thevertebrate. One important embodiment of the invention is a process forthe treatment of muscular dystrophy wherein said polynucleotideoperatively codes for dystrophin. Preferably, the composition isintroduced into the muscle tissue.

[0034] In a second aspect, the present invention relates to the use ofinterleukin-10 (IL-10) for the preparation of a therapeutic compositionfor introducing a polynucleotide into a cell. It was furthermoresurprisingly found that the addition of interleukin-10 when transfectinga polynucleotide into vertebrate tissue leads to a dramatic improvementof the transfection efficiency. In particular, it was surprisingly foundthat if the polynucleotide is injected together with an interleukin-10,e.g., into muscular tissue, the transfection is not only improved in thesurrounding of the injection site but also in other areas of the muscle.Thus, the present invention preferably relates to the use ofinterleukin-10 for the preparation of a pharmaceutical composition foran improved introduction of a polynucleotide into a cell. The term“improved introduction” in the scope of the present invention means, inthis regard, a more efficient uptake of a polynucleotide by cells wheninterleukin-10 is present compared to an introduction performed withoutinterleukin-10. This can be determined by comparing the amount of thepolynucleotide taken up without the use of interleukin-10 and comparingthis amount with the amount taken up by the cells when usinginterleukin-10 under the same experimental conditions. Preferably, theimproved introduction can be determined by a higher amount of expressionof the polynucleotide transferred into the cells when usinginterleukin-10 in comparison to a situation where no interleukin-10 isused.

[0035] Preferably, an improved introduction of the polynucleotide intothe cell means that the uptake of the polynucleotide by cells is notonly improved at the site of administration of the polynucleotide andinterleukin-10 but is also improved in neighboring cells. Thetherapeutic compositions according to the second aspect of the presentinvention are particularly useful for the delivery of polynucleotides tocells or tissues of a subject in the scope of a gene therapeutic methodbut are not limited to such use. The term “gene therapy method” ispreferably understood as a method for the introduction of apolynucleotide into cells either in vivo or by introduction into cellsin vitro followed by re-implantation into a subject. “Gene therapy” inparticular concerns the case where the gene product is expressed in atarget tissue as well as the case where the gene product is excreted,especially into the blood stream.

[0036] In the scope of the present invention the term “introduction”means the transfer of the polynucleotide into a cell (transfection).

[0037] Since its discovery in 1990, interleukin-10 (IL-10), which is apleiotropic hormone, has been implicated as an important regulator offunction of the immune system (Moore et al., Annu. Rev. Immunol. 11(1993), 165-190). In the scope of the present invention IL-10 isunderstood to be a cytokine that inhibits cell-mediated immunity andinflammation while promoting humoral responses. Naturally, the cytokineIL-10 is produced by Th0 and Th2 cells, β-lymphocytes,monocytes/macrophages, keratinocytes and bronchial epithelial cells(reviewed by Demoly et al., Gene Ther. 4 (1997), 507-516). In the scopeof the present invention IL-10 is preferably understood to have at leastone of the following characteristics. It activates both proliferationand viability of B lymphocytes and mast cells, increases E-selectinexpression of endothelial cells and neutrophil accumulation at the siteof inflammation (Vora et al., J. Exp. Med. 184 (1996), 821-829).Moreover, it increases Bcl-2 expression and survival of hematopoieticprogenitor cells (Weber-Nordt et al., Blood 88 (1996), 2549-2548). Onthe other hand, IL-10 presents also many other properties:

[0038] it enhances resolution of inflammation by promoting clearance ofrecruited neutrophils through apoptosis (Cox, Am. J. Physiol. 271(4Pt 1) (1996), L566-L571);

[0039] it downregulates monocyte/macrophages, Langerhans and dendriticcell functions (increases bacteria intracellular survival, lowerscytokine synthesis, oxygen free radical genesis and antigenpresentation);

[0040] it indirectly prevents antigen-specific T-cell activation, whichis associated with inhibition of MHC class 11 antigen presentation andaccessory cell functions of presenting cells to T cells and NK cells(Powrie and Coffman, Res Immunol. 144 (1993), 639-643; Moore et al.,Annu. Rev. Immunol. 11 (1993), 165-190; Murray et al., J. Immunol. 158(1997), 315-321);

[0041] it inhibits Th1 lymphocyte and neutrophil expansion and thesynthesis of their cytokines (IL-2, IFN-γ, IL-3, TNF, GM-CSF) and alsoeosinophil survival and cytokine production (GM-CSF, TNF-α, IL8);

[0042] it is required to prevent immune hyperactivity during infectionwith various agents (parasites, bacteria, viruses) (Hunter et al., J.Immunol. 158 (1997), 3311-3116);

[0043] it indirectly suppresses tumor growth and certain tumors byinhibiting infiltration of macrophages which may provide tumor growthpromoting activity (Richter et al., Cancer Res. 53 (1993), 4134-4137).Fibrinogen (a potential marker of vascular disease) is alsodownregulated by IL-10 (Vasse et al., Br. J. Haematol. 93 (1996),955-961).

[0044] These overall properties together with its good tolerability ledto the conviction that IL-10 has great potential therapeutic utility inthe treatment of diseases, such as chronic inflammation, autoimmunediseases, transplant rejection, graft-versus-host disease, sepsis (deVries, Ann. Med. 27 (1995), 537-541), asthma (Demoly et al., Gene Ther.4 (1997), 507-516) and cancer (Richter et al., Cancer Res. 53 (1993),4134-4137).

[0045] Accordingly, many pharmaceutical applications of interleukin-10have already been described such as for example:

[0046] pretreatment with -rhIL-10 of patient which reducesendotoxin-induced febrile responses, cytokine responses, and granulocyteaccumulation in human lungs (Pajkrt et al., J. Immunol. 158 (1997),3971-3977). In vivo topical application of IL-10 induces down-regulationof preinflammatory cytokine secretion both systemically and locally inpatients with inflammatory bowel disease (Schreiber et al.,Gastroenterology 108 (1995), 1434-1444) and psoriasis (Michel et al.,Inflamm. Res. 46 (1997), 32-34) In the later example, the IL-10 inducervitamin D3 (and its analogues) attracted interest as new therapeuticagents;

[0047] IL-10 was also shown to attenuates both local and distant organinjury in lung and skeletal muscle (Engles et al., J. Surg. Res. 69(1997), 425-428);

[0048] prolongation of allograft survival can be achieved through genetransfer of gene encoding TGF-β or IL-10 inducing a transient expressionof the cytokines within allografts and allowing local immunosuppressionwhile avoiding the systemic toxicity of conventional immunosuppression(Qin et al., Transplantation 59 (1995), 809-816; Fabrega et al.,Transplantation 62 (1996), 1866-1871);

[0049] IL-10 suppressive action (alone or in combination with IL-4 orTGF-b) on inflammatory or immunostimulant cytokines led to applicationsfor autoimmune diseases such as diabetes (Moritani et al., J. Clin.Invest. 98 (1996) 1851-1859), rheumatoid arthritis (Sugiyama et al., J.Rheumatol. 22 (1995), 2020-2026), systemic lupus erythematosus ormultiple sclerosis (Salmaggi et al., J. Neurol. 243 (1996), 13-17).IL-10 is also produced by Schwann cells that provide a constitutiveimmunosuppressant system for the peripheral nervous system (Jander etal., J. Neurosci. Res. 43 (1996), 254-259). IL-10 may play a role inglial cell differentiation and proliferation (Zocchia et al., NeurochemInt. 30 (1997), 433-439);

[0050] IL-10 has been shown to suppress cytokine production andinflammation in various animal models of microbial infection orirritation of various tissues (i.e. digestive track (Herfarth et al.,Gut 39 (1996), 836-845); skin (Berg et al., J. Exp. Med. 182 (1995),99-108); eyes (Hayashi et al., Graefes Arch. Clin. Exp. Ophtalmol. 234(1996), 633-636) and lung (Grünig et al., J. Exp. Med. 185 (1997),1089-1099). Intratracheal injection of IL-10 several minutes beforeallergenic provocation in ovalbumin-sensitized rats significantlyinhibits the inflammation process (reviewed by Demoly et al., Gene Ther.4 (1997), 507-516).

[0051] The term “interleukin-10 (IL-10)” as used herein, preferablymeans a polypeptide having the amino sequence disclosed in Vieira et al.(Proc. Natl. Acad. Sci., 88 (1991), 1172-1176) or in Kim et al. (J. ofImmunology 148 (1992) 3618-3623) or a variant of said polypeptide.

[0052] An interleukin-10 (IL-10) variant as referred to herein is apolypeptide substantially homologous to a sequence of a native mammalianIL-10 but that has an amino acid sequence different from said nativemammalian IL-10 polypeptide because of an amino acid deletion, addition,insertion or substitution. Variants may comprise conservativelysubstituted sequences, meaning that a given amino acid residue isreplaced by a residue having similar physiochemical characteristics.Examples of conservative substitutions include substitution of onealiphatic residue for another, such as lie, Val, Leu or Ala for oneanother, or substitutions of one polar residue for another, such asbetween Lys and Arg, Glu and Asp, or Gln and Asn. Other suchconservative substitutions, for example, substitutions of an entireregion having similar hydrophobicity characteristics, are well known inthe art. Naturally occurring IL-10 variants are also encompassed by theinvention.

[0053] The term IL-10 as used herein also means IL-10 as derived from avariety of mammalian species, including, for example, human, simian,rabbit, bovine, porcine or murine. IL-10 can be produced by recombinanttechnology. IL-10 is supplied, for example, by Sigma.

[0054] In a preferred embodiment of the use according to the secondaspect of the present invention the prepared therapeutic composition isin a form for administration into a vertebrate tissue. These tissuesinclude those of muscle, skin, brain, lung, liver, spleen, bone marrow,thymus, heart, lymph, bone, cartilage, pancreas, kidney, gall bladder,stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye,gland, connective tissue, blood, tumor etc. Cells where the improvedtransfection of a foreign polynucleotide would be obtained are thosefound in each of the listed target tissues (muscular cells, airwaycells, hematopoietic cells, etc.). The administration may be made byintradermal, subdermal, intravenous, intramuscular, intranasal,intracerebral, intratracheal, intraarterial, intraperitoneal,intravesical, intrapleural, intracoronary or intratumoral injection,with a syringe or other devices. Transdermal administration is alsocontemplated, as are inhalation or aerosol administration.

[0055] In a preferred embodiment, the therapeutic composition is for theintroduction into muscle tissue, more preferably, by intramuscularinjection routes.

[0056] In another preferred embodiment of the second aspect of thepresent invention, the use of interleukin-10 for the preparation of atherapeutic composition for improving transfection of a polynucleotideinto a cell is provided wherein said therapeutic composition isadministered independently from a second administration consisting inadministration of a composition containing at least one polynucleotide.According to the present invention, the first administration can be doneprior to, concurrently with or subsequent to the second administration,and vice-versa. The therapeutic composition administration and secondadministration can be performed by different or identical deliveryroutes (systemic delivery and targeted delivery, or targeted deliveriesfor example). In a preferred embodiment, each should be done into thesame target tissue and most preferably by injection.

[0057] In a further preferred embodiment of the use according to thepresent invention, the therapeutic composition further comprises atleast one polynucleotide. In a particularly preferred embodiment, thepolynucleotide which is contained in the composition, contains and iscapable of functionally expressing a gene in said cell.

[0058] The polynucleotide may be a DNA or RNA, single or doublestranded, linear or circular, natural or synthetic, modified or not (seeU.S. Pat. No. 5,525,711, U.S. Pat. No. 4,711,955 or EP-A 302 175 formodification examples). It may be, inter alia, a genomic DNA, a cDNA, anmRNA, an antisense RNA, a ribosomal RNA, a ribozyme, a transfer RNA orDNA encoding such RNAs. “Polynucleotides” and “nucleic acids” aresynonyms in the scope of the present invention. The polynucleotide mayalso be in the form of a plasmid or linear polynucleotide which containsat least one expressible sequence of nucleic acid that can generate apolypeptide, a ribozyme, an antisense RNA or another molecule ofinterest upon delivery to a cell. The polynucleotide can also be anoligonucleotide which is to be delivered to the cell, e.g., forantisense or ribozyme functions. The polynucleotide according to thesecond aspect of the present invention should preferably be understoodas a naked polynucleotide (Wolff et al., Science 247 (1990), 1465-1468)or as a polynucleotide associated or complexed with a viral polypeptideor a cationic compound or with any component which can participate inthe uptake of the polynucleotide into the cells (see Ledley, Human GeneTherapy 6 (1995), 1129-1144 for a review). Both DNA or RNA can bedelivered to cells to form therein a polypeptide of interest.Preferably, the polynucleotide present in the therapeutic composition isin the form of plasmid DNA. If the polynucleotide contains the propergenetic information, it will direct the synthesis of relatively largeamounts of the encoded polypeptide. When the polynucleotide delivered tothe cells encodes an immunizing polypeptide, the use according to theinvention can be applied to achieve improved and effective immunityagainst infectious agents, including intracellular viruses, and alsoagainst tumor cells. The genetic information necessary for expression bya target cell comprise all the elements required for transcription ofsaid DNA into mRNA and for translation of mRNA into polypeptide.Transcriptional promoters suitable for use in various vertebrate systemsare well known. For example, suitable promoters include viral promoterslike RSV, MPSV, SV40, CMV or 7.5 k, vaccinia promoter, induciblepromoters, etc. The polynucleotide can also include intron sequences,targeting sequences, transport sequences, sequences involved inreplication or integration. Said sequences have been reported in theliterature and can be readily obtained by those skilled in the art. Thepolynucleotide can also be modified in order to be stabilized withspecific components as spermine.

[0059] According to the invention, the polynucleotide can be homologousor heterologous to the target cells into which it is introduced.Advantageously said polynucleotide encodes all or part of a polypeptide,especially a therapeutic or prophylactic polypeptide. A polypeptide isunderstood to be any translational product of a polynucleotideregardless of size, and whether glycosylated or not, and includespeptides and proteins. Therapeutic polypeptides include as a primaryexample those polypeptides that can compensate for defective ordeficient proteins in an animal or human organism, or those that actthrough toxic effects to limit or remove harmful cells from the body.They can also be immunity conferring polypeptides which act asendogenous immunogens to provoke a humoral or cellular response, orboth. Examples of polypeptides encoded by the polynucleotide areenzymes, hormones, cytokines, membrane receptors, structuralpolypeptides, transport polypeptides, adhesines, ligands, transcriptionfactors, traduction factors, replication factors, stabilization factors,antibodies, more especially CFTR, dystrophin, factors VIII or IX, E6 orE7 from HPV, MUC1, BRCA1, interferons, interleukin (IL-2, IL4, IL6,IL-7, IL-12, GM-CSF (Granulocyte Macrophage Colony Stimulating Factor),the tk gene from Herpes Simplex type 1 virus (HSV-1), p53 or VEGF. Thepolynucleotide can also code for an antibody. In this regard, antibodyencompasses whole immunoglobulins of any class, chimeric antibodies andhybrid antibodies with dual or multiple antigen or epitopespecificities, and fragments, such as F(ab)₂, Fab′, Fab including hybridfragments and anti-idiotypes (U.S. Pat. No. 4,699,880).

[0060] According to the second aspect the invention relates to acomposition for the introduction of a polynucleotide into a cell, saidcomposition comprising at least one polynucleotide and interleukin-10.Polynucleotide and interleukin-10 components are defined as above.

[0061] According to the present invention, the amount of interleukin-10in the compositions ranges preferably between about 0.001 to about 1 μg,preferably from about 0.01 to about 0.1 μg of interleukin-10.

[0062] In another preferred embodiment, the polynucleotide which iscontained in the composition, contains and is capable of functionallyexpressing, a gene in a cell, preferably in a vertebrate cell. Oneparticularly preferred embodiment of the invention is a compositionwherein said polynucleotide is naked. Nevertheless, the polynucleotidecomprised in said composition can also be associated with viralpolypeptides, or complexed with cationic components, more particularlywith cationic lipids. In general, the concentration of polynucleotide inthe composition is from about 0.1 μg/ml to about 20 mg/ml.

[0063] In a further preferred embodiment the composition furthercomprises at least one component selected from the group consisting ofchloroquine, protic compounds such as propylene glycol, polyethyleneglycol, glycerol, ethanol, 1-methyl L-2-pyrrolidone or derivativesthereof, aprotic compounds such as dimethylsulfoxide (DMSO),diethylsulfoxide, di-n-propylsulfoxide, dimethylsulfone, sulfolane,dimethyl-formamide, dimethylacetamide, tetramethylurea, acetonitrile orderivatives.

[0064] In another preferred embodiment the composition according to theinvention can be used in a method for the therapeutic treatment ofhumans or animals. In this particular case, the composition according tothe second aspect of the invention may also comprise a pharmaceuticallyacceptable injectable carrier (for examples, see Remington'sPharmaceutical Sciences, 16^(th) ed. 1980, Mack Publishing Co). Thecarrier is preferably isotonic, hypotonic or weakly hypertonic and has arelatively low ionic strength, such as provided by a sucrose solution.Furthermore, it may contain any relevant solvents, aqueous or partlyaqueous liquid carriers comprising sterile, pyrogen-free water,dispersion media, coatings, and equivalents, or diluents (e.g.,Tris-HCl, acetate, phosphate), emulsifiers, solubilizers or adjuvants.The pH of the pharmaceutical preparation is suitably adjusted andbuffered.

[0065] Furthermore, the present invention also relates to a process forintroducing a polynucleotide into cells wherein said process comprisescontacting said cells with at least one composition according to theinvention. This process may be applied by direct administration of saidcomposition to cells of the animal in vivo, or by in vitro treatment ofcells which can be extracted from the animal and then re-introduced intothe animal body (ex vivo process). According to the practice of theinvention, targeted “cells” and “in vivo administration route” aredefined as above described.

[0066] The present invention also relates to a process for introducing apolynucleotide into cells wherein said process comprises contacting thecells with said polynucleotide prior to, concurrent with or subsequentto contacting them with interleukin-10. “Interleukin-10”,“polynucleotide” and the target cells are defined as above.

[0067] Preferably, muscle is used as a site for the delivery andexpression of a polynucleotide in a number of therapeutic applicationsbecause animals have a proportionately large muscle mass which isconveniently accessed by direct injection through the skin. Accordingly,in a preferred case, the invention concerns a process for introducing apolynucleotide, preferably in naked form, into muscle cells in vivo,comprising the steps of administering in vivo at least a polynucleotideand interleukin-10, preferably intramuscularly, whereby thepolynucleotide is introduced into muscle cells of the tissue. Thepolynucleotide may encode a therapeutic polypeptide that is expressed bythe muscle cells and eventually secreted into the blood stream after thecontacting step to provide therapy to the vertebrate. Similarly, it mayencode an immunogenic polypeptide that is expressed by the muscle cellsafter the contacting step and which generates an immune response,thereby immunizing the vertebrate. One important embodiment of theinvention is a process for the treatment of muscular dystrophy whereinsaid polynucleotide operatively codes for dystrophin. Preferably, thecomposition is introduced into the muscle tissue.

[0068] The invention has been described in an illustrative manner, andit is to be understood that the terminology which has been used isintended to be in the nature of words of description rather than oflimitation. Obviously, many modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the claims, the invention maybe practiced otherwise than as specifically described.

[0069]FIG. 1: Effect of DNAse inhibitors on pTG11033 intramusculartransfection. Luciferase activity of mouse right and left tibialisanterior muscles measured 7 days after injection with 25 μg plasmidadded with NaCl 0.9% buffer (Control, empty bar) or with 10 μg ofG-actin (G-actin, full black bar). Bars are means of RLU (Relative LightUnit) per minute per mg proteins +/−s.e.m. of 8 determinations.

[0070]FIG. 2: Combinations of adjuvants including G-actin to improveintramuscular transfer of the luciferase-plasmid (pTG11033).

[0071] Bars are means of RLU per minute per mg proteins +/−s.e.m of 8determinations. Luciferase activity was measured 7 days after plasmidinjection into C57BL/10 mice (4 mice per group) added with either NaCl0.9% (empty bars) or different combinations of adjuvants (black bars),NaCl 0.9%, IL-10 0.1 μg, G-actin 10 μg, DMSO 10% final, G-actin+IL-10,or DMSO+G-actin+IL-10, G-actin+DMSO, DMSO+IL-10.

[0072]FIG. 3: Dose/Response effect of G-Actin. Luciferase activity ofright and left tibialis anterior muscles of 4 mice per group, 7 daysafter injection with plasmid added with NaCl 0.9% (empty bar) ordifferent doses of G-actin (full black bars). Bars are means of RLU perminute per mg proteins +/− s.e.m. of 8 determinations.

[0073]FIG. 4: Effect of Interleukin-10 (IL-10) on pTG11033 intramusculartransfection. Luciferase activity of mouse right and left tibialisanterior muscles measured 7 days after injection with 25 μg plasmidadded with NaCl 0.9% buffer (Control, empty bar) or with either 0.01 or0.1 μg IL-10. Bars are means of RLU (Relative Light Unit) per minute permg proteins +/−s.e.m. of 12 determinations.

[0074]FIG. 5: Detection of human IFN-β in the serum of mice (A: SCID orB: C57BL/10) injected intramuscularly with pTG13102 with our withoutadjuvants mixture (G-actin, IL-10, DMSO).

[0075]FIG. 6: Luciferase activity in luciferase-plasmid injected mousemuscles is increased by G-actin.

The following examples illustrate the invention. MATERIAL AND METHODS

[0076] The following materials and methods are used in the examples.

[0077] 1. Plasmid/interleukin-10 Composition IntramuscularAdministration

[0078] Plasmids are prepared according to Bischoff et al. (AnalyticalBiochemistry 254 (1997), 69-81). The tested interleukin-10s were mixedwith the plasmid preparation (pTG11033: CMV promoter, β-globin intron,luciferase cassette; pTG11025: CMV promoter, β-globin intron, dystrophincassette; both diluted in 0.9% NaCl), prior to intramuscular injection.25 μg of plasmid are injected per muscle in 5 to 10 week-old C57Bl/10 ormdx mice. The 2 tibialis anterior (right and left) muscles were injected(each muscle was considered as a sample, which means number of samplesper condition=2×number of mice per condition).

[0079] 2. Muscle Biopsies

[0080] One week after injection of the composition, mice were killed andthe tibialis anterior muscles were retrieved and frozen. Based on theinjected vector, either luciferase activity was determined in muscleextracts, or dystrophin expression was evaluated byimmunohistochemistry.

[0081] 3. Luciferase Measurement

[0082] Luciferase activity was quantified using a conventionalmeasurement kit (Luciferase Assay System, Promega). Briefly, muscleswere ground separately and diluted in 200 μl of reporter lysis buffer(Promega). 10 μl-samples were placed in the 96 well-plates and mixedwith 100 μl of substrate. Luciferase activity was expressed as number ofRLU emitted per minute.

[0083] 4. Protein Determination

[0084] Proteins were measured on 10 μl samples using a VCA Protein AssayKit (Pierce).

[0085] 5. Dystrophin Immunohistochemistry

[0086] Muscle samples were frozen in liquid nitrogen-cooled isopentaneand stored at −80° C. Immunofluorescence microscopy usinganti-dystrophin antibody was performed as follows: serial cryostattransverse sections (5-8 μm) of unfixed muscles were prepared on glassslices, dipped in PBS buffer with 1% mouse serum and incubated for 30min at room temperature, for saturation of the non specific bindingsites. After rinsing (3 times, 5 min) in PBS buffer, slices were dippedin PBS buffer containing {fraction (1/500)} dilution of anti-dystrophinmonoclonal antibody (MANDRA-1, Sigma) and incubated for 90 min at roomtemperature. Slices were rinsed 3 times for 5 min in PBS, then incubatedfor 30 min at room temperature with biotin-F(ab′)₂ goat anti-mouse IgG(H+L) diluted {fraction (1/500)} in PBS. After rinsing (3 times, 5 minin PBS), preparations were incubated 30 min at room temperature with{fraction (1/2000)} Streptavidin-FITC (1 mol Streptavidin/4 mol Biotin).Slices were then rinsed and mounted with Mowiol for microscopicevaluation.

[0087] 6. IFN-β Titration

[0088] The ELISA titers were challenged with a biological test. Thebiological activity of human IFN-β corresponded to the protective effectof IFN-β against VSV infection of WISH cells. The titers were determinedaccording to the dilution of the sample which protects 50% of the cellsfrom VSV cytopathic effect. IFN titers were equivalent between ELISA andbiological assays, indicating that the IFN detected corresponds to afunctional protein. Following standard IFNs were used to calibrate theassays:

[0089] human IFN-β NIH (Stock TG 1995 stored at −20° C.)

[0090] human IFN-β from the ELISA kit.

[0091] The ELISA test was performed according to the manufacturer'srecommendation.

EXAMPLE 1 Effect of DNAse Inhibitor on PTG11033 IntramuscularTransfection

[0092] In this example, 10 μg of G-actin were added to pTG11033 (25μg/muscle) in a total volume of 30 μl to be injected. 4 C57BL/10 adultmice (male and female) per group have been injected in tibialis anteriormuscles. The control experiment is performed according to the samecondition except that no G-actin is added. The results are presented inFIG. 1. They show that the addition of G-actin leads to a significantincrease of intramuscular transfection of the plasmid in muscular cells.This example shows that G-actin increases significantly (about 12 timesin the present example) gene transfer into skeletal muscle as evidencedby luciferase activity measurement of the injected muscles 7 days afterplasmid administration.

EXAMPLE 2

[0093] Combinations of Adjuvants including G-actin to Improve (i.m.)Transfer of Dystrophin-plasmid (pTG11025)

[0094] It was tried to enhance the observed improvement by adding othercomponents to the composition of the invention. In this example, 6 mdxmice were injected (final volume injected: 35 μl) with pTG11025 plasmidpreparation added with:

[0095] (1) NaCl 0.9%;

[0096] (2) IL-10 0.1 μg;

[0097] (3) DMSO 10% final;

[0098] (4) G-actin 10 μg;

[0099] (5) G-actin 10 μg+IL-10 0.1 μg; or

[0100] (6) DMSO 10% final+G-actin 10 μg+IL-10 0.1 μg

[0101] In these experiments, notexin-induced necrosis-regeneration wascarried out 3 days prior to plasmid injection (Lefaucheur et Sebille,Neuromuscul. Disord. 5 (1995), 501-509).

[0102] Tibialis anterior muscles were collected 7 days after injection,and histological analysis of the transfected tibialis anterior musclehave been conducted.

[0103] The results (Table I) show the following order of efficiency:6>4>5>3>2>1 TABLE I Number of dystrophin-positive fibers per Testedmolecules cryosection DMSO + G-actin + IL-10  89.3 +/− 19.7 G-actin 71.0+/− 6.0 G-actin + IL-10 44.5 +/− 6.9 DMSO 37.8 +/− 5.5 IL-10 21.7 +/−1.9 NaCl  3.3 +/− 0.7

[0104] Values are mean +/− sem of up to 4 determinations par muscle(obtained from the serial sections of the whole muscles).

[0105] In this experiment the best condition (DMSO+G-actin+IL-10) led toaround 15% of dystrophin-positive fibers in the mdx tibialis anteriormuscles. Interestingly, histological analysis shows that those positivefibers were not localized to a fraction of the muscle (i.e. along theneedle track), but homogeneously in the transversal sections. Example 2(as well as example 3) show that combinations of G-actin and othercompounds may act synergistically on gene transfer.

[0106] In a second experiment mdx mice were 3 times injectedintramuscularly with pTG11025 (dystrophin) plus DMSO, G-actin and IL-10after notexin-induced muscle regeneration. Plasmid-adjuvant injectionswere repeated 3 times in raw on a daily basis and dystrophin expression(immunohistochemistry) was evaluated 7 days after the last injection. Inthis case up to 20% of dystrophin-positive fibers were found in theinjected muscles.

EXAMPLE 3 Combinations of Adjuvants including Interleukin-10 (IL-10) orG-actin to Improve Intracellular Transfer of Luciferase-plasmid(pTG11033)

[0107] It was tried to enhance the observed improvement by adding othercomponents to the composition of the invention. In this example, micewere injected (final volume injected: 35 μl) with a pTG11033(luciferase) plasmid preparation to which the following was added:

[0108] 1) NaCl 0.9%

[0109] 2) IL-10 0.1 μg,

[0110] 3) G-actin 10 μg,

[0111] 4) DMSO 10% final

[0112] 5) G-actin 10 μg+IL-10 0.1 μg,

[0113] 6) DMSO 10% final+G-actin 10 μg+IL-10 0.1 μg

[0114] 7) G-actin 10 μg+DMSO, 10%

[0115] 8) DMSO 10%+IL-10

[0116] Luciferase activity was measured 7 days after injection of thecomposition in C57BL/10 mice (4 mice per group).

[0117] The data show (FIG. 2) that injection of naked DNA in accordancewith the present invention (intramuscularly) produced improvedexpression in muscle which is not limited to the injection site. Thus,one of the fundamental differences between the present invention and theprior art methods is that the present invention results in an increasednon-localized gene expression in muscle cells and thus provides thepossibility for improving gene expression which is not possible withprior art methods. Furthermore, because of the need for fewer injectionsfor equivalent efficiency, application of the present invention islikely to be better tolerated by patients.

EXAMPLE 4 Dose/Response Effect of G-Actin

[0118] 25 μg of plasmid DNA (pTG11033 preparation at 2 mg/ml in 0.9%NaCl) was added with various dilutions (in 0.9% NaCl) of G-actin atfinal concentrations ranging from 0.01 to 10 μg per 30 μl (finalvolume), 4 mice per condition. Injections were performed in both rightand left tibialis anterior. Luciferase activity was measured in musclesthat were collected 7 days after plasmid injection. As shown by FIG. 3,luciferase activity is increased in the muscle that has been injectedwith plasmid added with G-actin even at low concentrations. Maximaleffect seems to be obtained at G-actin concentrations of 0.1 to 1 μg/25μg plasmid DNA. Bars are mean +/−sem of 8 values per condition.

EXAMPLE 5 Expression of a Gene Encoding a Secreted Protein afterIntramuscular Injection of PTG13102 in SCID and C57BL/10 Mice

[0119] The expression of an IFN-β encoding plasmid pTG13102 was examinedafter injection into SCID and C57BL/10 mice. Said plasmid is based onthe backbone pTG11022 (kanamycin, pCMV, HMG intron, SV40pA, CER andcarrying the human IFN-β cDNA). Thus, this plasmid construct shows thesame backbone as pTG11033 but the gene encodes the human interferon-betaand allows high level production of hulFN-β in vitro (about 50,000IU/ml). The hulFN-β plasmid was validated in vitro (calcium phosphatetransfection) on the mouse muscle cell line C2C12. hulFN-β was measuredusing a standardized ELISA kit (Fujirebio). G-actin from porcine muscle(purchased from Sigma, L'lsle d'Abeau Chesnes, France) was diluted at 5μg/μl in distilled water and stored at −20° C. until use.

[0120] Plasmid pTG13102 was injected in adult C57BL/10 and SCID mice inthe presence of the following adjuvants: G-actin, IL-10, or DMSO. 25 μgplasmid was injected in each of the right and left tibialis anterior(TA) and quadriceps muscles (Quadr.). Six groups of 3 mice were injected4 times into the right and left TA and Quadr. muscles with 25 μg ofpTG131102 in NaCl 0.9% or with a mixture of 10 μg G-actin, 0.1 mM MgCl₂and/or DMSO (10% final). The total volume injected per muscle was 30 μl.Prior to plasmid administration, muscles were treated 3 days byinjecting 3 ng/25 μl of notexine in order to induce muscle regeneration(which follows the notexin-induced necrosis). Blood samples were takenat various time points. At day 7 and day 14 after plasmid injection,mice were sacrificed and their muscles were dissected. The muscles werecollected at the end of the experiment, frozen and grinded. Grindedsamples were then extracted using a PBS buffer (600 μl and 400 μl volumefor tibialis and quadriceps, respectively). The supernatants were thenused for human IFN-β measurement.

[0121] Human IFN-β was detected in sera of both SCID (FIG. 5A) andC57BL/10 mice (FIG. 5B) for at least 2 weeks. The similar blood levelsof human IFN-β found in both SCID and C57BL/10 indicate that theimmunocompetent mice (the C57BL/10 mice) are equally transfected thanimmunodepressed animals (SCID mice).

[0122]FIG. 5A also demonstrates that higher IFN levels are observed whenthe plasmid is injected with an adjuvant, while very few if no IFN isexpressed in the case of adjuvant-free injected plasmid.

[0123] Moreover, the following table shows also that there is a goodcorrelation between IFN levels found in muscles and the correspondingsera (Table 1). TABLE 1 human IFN-β levels found in individual musclesand sera Mouse # (day IU/muscle IU/ml serum post injection) INJECTIONLeft TA Right TA Right Q Left Q Day 7 Day 14 SCID 1 pTG13102 5 4 1.2 0.32 pTG13102 1 2 0.15 3 pTG13102 8 1 0.35 (day 7) mean 4.67 2.33 1.20 0.27sem 2.03 0.88 0.06 4 pTG13102 + adjuvants 27 36 1.02 2.3 5 pTG13102 +adjuvants 0 24 3.9 3.4 6 pTG13102 + adjuvants 23 11 1.2 1 (day 7) mean16.67 23.67 1.02 2.55 2.23 sem 8.41 7.22 0.69 7 pTG13102 + adjuvants 636 1 3.6 1.33 8 pTG13102 + adjuvants 9 2 0.6 0.35 9 pTG13102 + adjuvants20 0 0 1.62 1.6 0.9 (day 14) mean 11.67 12.67 0.50 1.62 1.93 0.86 sem4.26 11.68 0.88 0.28 C57BL10 13 pTG13102 + adjuvants 9 7 1.2 14pTG13102 + adjuvants 0 22 3.3 15 pTG13102 + adjuvants 47 4 1.5 (day 7)mean 18.67 11.0 2.00 sem 14.40 5.57 0.66 16 pTG13102 + adjuvants 17 692.1 15 5.7 17 pTG13102 + adjuvants 18 0 1.5 1.4 18 pTG13102 + adjuvants0 1 0.3 0 (day 14) mean 11.67 23.33 2.10 5.60 2.37 sem 5.84 22.84 4.711.71 non injected muscle 0 0 non injected muscle 0 0 mean 0 0 sem 0 0

EXAMPLE 6 Expression of a Reporter Gene Encoding Luciferase afterIntramuscular Injection into C57BL/10 Mice

[0124] The gene expression of a reporter gene encoding luciferase in thepresence or absence of G-actin was tested. For this purpose, themolecule(s) being tested was/were injected intramuscularly together witha plasmid preparation of pTG11033 (pCMV-luciferase, same backbone as forpTG13102) in 5 to 10 week old C57BL/10 mice. The preparation wasinjected into both right and left TA muscles (25 μg plasmid, totalvolume 30 μl).

[0125] Four mice per condition were used. Due to the injections into theright and left tibialis of each mouse 8 samples per condition wereobtained. The highest and lowest values were discarded, thus 6 valuesper condition remained.

[0126] One week after injection of the vectors, the mice were killed andthe tibialis anterior muscles were retrieved and frozen. Luciferaseactivity was determined on muscle extracts. G-actin was diluted eitherin DMSO (10% final) or in distilled water at 5 μg/μl extemporarily andadded to the plasmid preparation alone or together with 10% final DMSOin the case of water-dissolved G-actin. As control, the plasmid alone(prepared in 0.9% NaCl) was injected. The data are shown in FIG. 6.

[0127] All adjuvants allowed increased luciferase activity in theinjected muscles. G-actin increases gene transfer whether the additiveis diluted in water or in DMSO.

EXAMPLE 7 IL-10 Increases Gene Transfer with a Plasmid Comprising theLuciferase Gene

[0128] 4 groups of 6 C57Bl/10 mice have been injected into the right andleft tibialis anterior muscle with 3 different compositions comprisingpTG11033 (25 μg/muscle) and 3 various doses of IL-10 (0, 0.1 and 0.01μg). The control experiment is performed according to the same conditionexcept that no IL-10 is added.

[0129] Final volume was 30 μl in NaCl 0.9% solution. The IL-10 used washuman recombinant IL-10 (Sigma).

[0130] The results are presented in FIG. 4 and show that intramuscularinjection of the luciferase plasmid pTG11033 in presence of IL-10 leadsto a dose-dependent increase of luciferase expression (factor of 3).

1. Use of a nuclease inhibitor for the preparation of a therapeuticcomposition for the introduction of a polynucleotide into a cell.
 2. Theuse of claim 1, wherein said nuclease inhibitor is a deoxyribonuclease(DNAse) inhibitor, preferably a DNAse I inhibitor.
 3. The use of claim2, wherein said nuclease inhibitor is G-actin or a fragment thereofcapable of inhibiting DNAse I activity.
 4. The use of claim 3, whereinsaid G-actin is of porcine, rabbit, bovine, simian, murine or humanorigin.
 5. The use of any one of claims 1 to 4, wherein said therapeuticcomposition is for administration into a vertebrate target tissue. 6.The use of claim 5, wherein said administration is made by intradermal,subdermal, intravenous, intramuscular, intranasal, intracerebral,intratracheal, intraarterial, intraperitoneal, intravesical,intrapleural, intracoronary or intratumoral injection.
 7. The use ofclaim 5, wherein said administration is made into the lung by inhalationor aerosol administration.
 8. The use of claim 5, wherein said targettissue is muscle.
 9. The use of any one of claims 5 to 8, wherein theadministration of the nuclease inhibitor is performed independently froma second administration consisting in administration of a compositioncontaining at least one polynucleotide into the same target tissue. 10.The use of claim 9, wherein the administration of the nuclease inhibitoris performed prior to said second administration.
 11. The use of any oneof claims 1 to 8, wherein said therapeutic composition further comprisesat least one polynucleotide.
 12. The use of any one of claims 1 to 11,wherein said polynucleotide contains a gene and is capable offunctionally expressing said gene in said cell.
 13. A composition forintroducing a polynucleotide into a cell, said composition comprising atleast one polynucleotide and at least one nuclease inhibitor.
 14. Thecomposition of claim 13, wherein said nuclease inhibitor is a DNAseinhibitor, preferably a DNAse I inhibitor.
 15. The composition of claim14, wherein said nuclease inhibitor is G-actin or a fragment thereofwith the capability to inhibit DNAse I activity.
 16. The composition ofclaim 15, wherein said G-actin or fragment thereof is porcine, rabbit,bovine or human origin.
 17. The composition of claim 15 or 16, whereinsaid composition contains between 4×10⁻⁵ and 4 μg, preferably between4×10⁻⁴ and 2 μg, and more preferably comprises between 4×10⁻³ and 4×10⁻¹μg of nuclease inhibitor per μg of DNA.
 18. The composition of any oneof claims 13 to 17, wherein the polynucleotide concentration ranges fromabout 0.1 μg/ml to about 20 mg/ml.
 19. The composition of any one ofclaims 13 to 18, wherein said polynucleotide contains a gene and iscapable of functionally expressing said gene in said cell.
 20. Thecomposition of claim 18, wherein said gene encodes all or part ofdystrophin or cystic fibrosis transmembrane conductance regulator (CFTR)polypeptides.
 21. The composition of any one of claims 13 to 20, whereinsaid cell is a vertebrate cell.
 22. The composition of any one of claims13 to 21, wherein said polynucleotide is naked.
 23. The composition ofany one of claims 13 to 21, wherein said polynucleotide is associatedwith viral polypeptides.
 24. The composition of any one of claims 13 to21, wherein said polynucleotide is complexed with cationic components,more preferably with cationic lipids.
 25. The composition of any ofclaims 13 to 24, wherein said composition further comprises at least onecomponent selected from the group consisting of chloroquine, proticcompounds such as propylene glycol, polyethylene glycol, glycerol,ethanol, 1-methyl L-2-pyrrolidone or derivatives, aprotic compounds suchas dimethylsulfoxide (DMSO), diethylsulfoxide, di-n-propylsulfoxide,dimethylsulfone, sulfolane, dimethylformamide, dimethylacetamide,tetramethylurea, acetonitrile or derivatives, cytokines, preferablyinterleukin 10 (IL-10).
 26. The composition of claim 25, wherein saidcomposition comprises 5-15% of DMSO and/or from about 0.001 to about 1μg preferably from about 0.01 to about 0.1 μg of IL-10.
 27. Thecomposition of any of claims 13 to 26 for use in a method for thetherapeutic treatment of the human or animal body.
 28. The compositionof claim 27, wherein said composition further comprises apharmaceutically acceptable injectable carrier.
 29. A process forintroducing a polynucleotide into cells wherein said process comprisescontacting said cells with at least one composition of any one of claims13 to
 28. 30. A process for introducing a polynucleotide into cellswherein said process comprises contacting the cells simultaneously orsubsequently with a nuclease inhibitor and the polynucleotide.
 31. Theprocess of claim 30, wherein the cells are first contacted with thenuclease inhibitor and subsequently with the polynucleotide.
 32. Use ofinterleukin-10 (IL-10) for the preparation of a therapeutic compositionfor the introduction of a polynucleotide into a cell.
 33. The use ofclaim 32, wherein said interleukin-10 is of human, simian, rabbit,bovine, porcine or murine origin.
 34. The use of claims 32 and 33,wherein said therapeutic composition is for administration into avertebrate target tissue.
 35. The use of claim 34, wherein saidadministration is made by intradermal, subdermal, intravenous,intramuscular, intranasal, intracerebral, intratracheal, intraarterial,intraperitoneal, intravesical, intrapleural, intracoronary orintratumoral injection.
 36. The use of claim 34, wherein saidadministration is made into the lung by inhalation or aerosoladministration.
 37. The use of claim 34, wherein said target tissue ismuscle.
 38. The use of any one of claims 34 to 37, wherein theadministration of the interleukin-10 is performed independently from asecond administration consisting in administration of a compositioncontaining at least one polynucleotide into the same target tissue. 39.The use of claim 38, wherein the administration of the interleukin-10 isperformed prior to said second administration.
 40. The use of any one ofclaims 32 to 37, wherein said therapeutic composition further comprisesat least one polynucleotide.
 41. The use of any one of claims 32 to 40,wherein said polynucleotide contains a gene and is capable offunctionally expressing said gene in said cell.
 42. A composition forintroducing a polynucleotide into a cell, said composition comprising anIL-10 and at least one polynucleotide.
 43. The composition of claim 42,wherein said interleukin-10 is of human, simian, rabbit, bovine, porcineor murine origin.
 44. The composition of claim 42 or 43, wherein saidcomposition contains between from about 0.001 to about 1 μg, preferablyfrom about 0.01 to about 0.1 μg of IL-10.
 45. The composition of any oneof claims 42 to 44, wherein the polynucleotide concentration ranges fromabout 0.1 μg/ml to about 20 mg/ml.
 46. The composition of any one ofclaims 42 to 45, wherein said polynucleotide contains a gene and iscapable of functionally expressing said gene in said cell.
 47. Thecomposition of claim 46, wherein said gene encodes all or part ofdystrophin or cystic fibrosis transmembrane conductance regulator (CFTR)polypeptides.
 48. The composition of any one of claims 42 to 47, whereinsaid cell is a vertebrate cell.
 49. The composition of any one of claims42 to 48, wherein said polynucleotide is naked.
 50. The composition ofany one of claims 42 to 49, wherein said polynucleotide is associatedwith viral polypeptides.
 51. The composition of any one of claims 42 to48, wherein said polynucleotide is complexed with cationic components,more preferably with cationic lipids.
 52. The composition of any ofclaims 42 to 51, wherein said composition further comprises at least onecomponent selected from the group consisting of chloroquine, proticcompounds such as propylene glycol, polyethylene glycol, glycerol,ethanol, 1-methyl L-2-pyrrolidone or derivatives, aprotic compounds suchas dimethylsulfoxide (DMSO), diethylsulfoxide, di-n-propylsulfoxide,dimethylsulfone, sulfolane, dimethylformamide, dimethylacetamide,tetramethylurea, acetonitrile or derivatives, cytokines different frominterleukin 10 (IL-10).
 53. The composition of claim 52, wherein saidcomposition further comprises 5-15% of DMSO.
 54. The composition of anyof claims 42 to 53 for use in a method for the therapeutic treatment ofthe human or animal body.
 55. The composition of claim 54, wherein saidcomposition further comprises a pharmaceutically acceptable injectablecarrier.
 56. A process for introducing a polynucleotide into cellswherein said process comprises contacting said cells with at least onecomposition of any one of claims 52 to
 55. 57. A process for introducinga polynucleotide into cells wherein said process comprises contactingthe cells with said polynucleotide prior to, concurrent with orsubsequent to contacting them with interleukin-10.
 58. The process ofclaim 57, wherein the cells are first contacted with the interleukin-10and subsequently with the polynucleotide.